Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. It is widely believed that receptor activation triggers the dissociation of heterotrimeric G proteins (Gαβγ) into two functional entities, Gα and Gβγ, for signaling. Gβγ signaling has become increasingly popular owing to its diverse downstream effectors and thus potential implications in numerous pathological disorders. Yet, the existence of receptor-induced Gβγ signal has not been rigorously proven in all scenarios because its downstream effectors overlap substantially with those regulated by the Gα subunit. Thus, in order to unambiguously observe Gβγ signals from an activated receptor, the function of its counterpart, Gα, has to be eliminated first. Given that Gβ...[ Read more ]

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. It is widely believed that receptor activation triggers the dissociation of heterotrimeric G proteins (Gαβγ) into two functional entities, Gα and Gβγ, for signaling. Gβγ signaling has become increasingly popular owing to its diverse downstream effectors and thus potential implications in numerous pathological disorders. Yet, the existence of receptor-induced Gβγ signal has not been rigorously proven in all scenarios because its downstream effectors overlap substantially with those regulated by the Gα subunit. Thus, in order to unambiguously observe Gβγ signals from an activated receptor, the function of its counterpart, Gα, has to be eliminated first. Given that Gβγ signals are frequently observed in a G_i-coupled system, this study aims to generate a Gα_i1 mutant that loses its ability to inhibit adenylyl cyclase (AC), its cognate effector, but is otherwise fully functional. A series of Gα_i1 chimeras was generated by replacing its putative effector-interacting domains with the homologous regions of Gα_t1. All Gα_i1/t1 chimeras failed to eliminate the constitutive inhibition from Gα_i1QL, whereas the constitutive activity of Gα_i1RC was abolished by some chimeras. Yet, these chimeras still retained the ability to inhibit AC upon receptor activation. Dose-response analysis on Gα_i1QL and Gα_i1RC suggested a differential inhibitory profile between the two commonly used constitutively active mutants. Besides, sequence comparison between inhibitory Gα_i/z identified an RNK motif on the α4/β6 loop of Gα_z as a potential determinant for AC inhibition. Asn-to-Arg mutation on this motif causes Gα_z to constitutively inhibit AC and interact with Ric-8A more efficiently. Collectively, these results indicated that the regions responsible for AC inhibition on Gα_i1 may be distinct from other members of the G_i family. The AC interaction domains on different Gα_i/z subunits may have subtle differences that have not yet been elucidated.